PROJECT SUMMARY Intraepithelial lymphocytes (IELs) are an abundant and heterogeneous population of T cells that reside at the intestinal epithelium, a critical environmental interface between the intestinal tract and the body core. IELs are tasked with maintaining tolerance to a high volume of harmless stimuli from food and commensal microbiota while providing protective immunity against ingested pathogens. Dysregulation of this balance leads to loss of intestinal barrier integrity, susceptibility to enteric infections, and inflammatory bowel diseases. However, many key aspects of IEL development, tissue residence, and function remain unclear, and the pursuit of these questions has been hampered by a lack of genetic targeting strategies for the majority of IEL subsets. We propose to undertake a trait mapping study using the recently established Diversity Outbred and Collaborative Cross mouse cohorts to identify novel genetic factors influencing IEL maturation and tissue maintenance. Recently, we performed quantitative trait loci analysis using these cohorts and identified 78 IEL-associated genomic regions. To determine which single nucleotide polymorphisms (SNPs) within IEL-associated loci are most likely to be causative, we will take a rigorous bioinformatics approach including functional characterization of SNPs, identification of candidate target genes, text mining for published association of candidate genes with relevant ontologies and cell or tissue types, and intestinal gene expression analysis. Further, for at least one candidate IEL-associated SNP identified we will use CRISPR/Cas9 gene editing to generate SNP mutants in C57BL/6 mice, with the goal of generating genetic models of high or low IEL frequency. These mice will be used to confirm biological relevance of candidate genetic elements in vivo, and will enable extensive future research into IEL function. The proposed work may promote understanding of how IELs contribute to gut homeostasis and suggest novel therapeutic targets for gut inflammatory diseases.